Project Summary Rejection remains a major hurdle to long-lasting transplant survival. From a clinical standpoint current immunosuppression protocols are fantastic in short-term transplant survival, but do little in promoting long- term graft outcomes. Thus, there is a pressing need to uncover effector programs that may emerge under broad immunosuppressive conditions that can mediate graft loss, with a goal of further improving long term transplant outcomes. We recently discovered that unlike other Th subsets, Th9 cells are robustly induced in an immunosuppressive milieu, suggesting that Th9 cells may be especially relevant to graft loss under conditions of immunosuppression. Th9 cells are induced in massive numbers when the default Th1/Th2 programs are inhibited and require the formation of super-enhancers at Il9 locus (not lineage specific transcription factors). Importantly, formation of Il9 super-enhancers requires OX40 signaling. Also, OX40 is remarkably potent in redirecting Foxp3+ Tregs to inflammatory Th9 cells. Based on the unexpected potency of OX40 in robust Th9 induction, as well as the unexpected mode of actions of OX40 stimulation, we propose to test a unifying hypothesis in this proposal, namely- suppression of the default T cell programs in an immunosuppressive milieu allows OX40 to promote alternative Th9 programs in triggering rejection of otherwise tolerant grafts. We propose 3 Aims in this proposal. Aim 1 focuses on identifying novel mechanisms by which OX40 promotes massive Th9 cells, examining formation, structure, and activities of super-enhancers at Il9 locus. Aim 2 investigates how OX40 redirects Foxp3+ Tregs to Th9 cells, focusing on the newly identified Foxp3 repressors in shutting down Foxp3 expression and examine whether Foxp3 may disrupt IL-9 super enhancer formations. Aim 3 examines the impact of Th9 cells in transplant survival, testing whether targeting super-enhancers or checkpoints in Th9 induction enables robust allograft tolerance. We believe that the proposed studies will unravel new mechanisms of tolerance resistance in transplant recipients, and findings from these studies will have broad impacts on other immune-mediated diseases as well, including cancer immunotherapies and autoimmune diseases.